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LincRNAs Act in the Circuitry Controlling Pluripotency and Differentiation (2011)

LincRNAs Act in the Circuitry Controlling Pluripotency and Differentiation (2011)

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Published by clementlawyer
Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.
Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have been functionally characterized, leading to debate about their biological role. To address this, we performed loss-of-function studies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on gene expression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns, comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in trans. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineage commitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genes are regulated by key transcription factors and that lincRNA transcripts bind to multiple chromatin regulatory proteins to affect shared gene expression programs. Together, the results demonstrate that lincRNAs have key roles in the circuitry controlling ES cell state.

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ARTICLE
lincRNAs act in the circuitry controlling pluripotency and differentiation
Mitchell Guttman
1,2
, Julie Donaghey
1
, Bryce W. Carey
2,3
, Manuel Garber
1
, Jennifer K. Grenier
1
, Glen Munson
1
, Geneva Young
1
,Anne Bergstrom Lucas
4
, Robert Ach
4
, Laurakay Bruhn
4
, Xiaoping Yang
1
, Ido Amit
1
, Alexander Meissner
1,5
*
, Aviv Regev
1,2
*
,John L. Rinn
1,5
*
, David E. Root
1
*
& Eric S. Lander
1,2,6
Although thousands of large intergenic non-coding RNAs (lincRNAs) have been identified in mammals, few have beenfunctionallycharacterized,leadingtodebateabouttheirbiologicalrole.Toaddressthis,weperformedloss-of-functionstudies on most lincRNAs expressed in mouse embryonic stem (ES) cells and characterized the effects on geneexpression. Here we show that knockdown of lincRNAs has major consequences on gene expression patterns,comparable to knockdown of well-known ES cell regulators. Notably, lincRNAs primarily affect gene expression in
trans 
. Knockdown of dozens of lincRNAs causes either exit from the pluripotent state or upregulation of lineagecommitment programs. We integrate lincRNAs into the molecular circuitry of ES cells and show that lincRNA genesareregulatedbykeytranscriptionfactorsandthatlincRNAtranscriptsbindtomultiplechromatinregulatoryproteinstoaffectsharedgeneexpressionprograms.Together,theresultsdemonstratethatlincRNAshavekeyrolesinthecircuitrycontrolling ES cell state.
The mammalian genome encodes many thousands of large non-coding transcripts
1
including a class of 
,
3,500 lincRNAs identifiedusing a chromatin signature of actively transcribed genes
2–4
. TheselincRNA genes have been shown to have interesting properties,including clear evolutionary conservation
2–5
, expression patterns cor-related with various cellular processes
2,6
and binding of key transcrip-tion factors to their promoters
2,6
, and the lincRNAs themselvesphysically associate with chromatin regulatory proteins
4,7
. Yet, itremains unclear whether the RNA transcripts themselves have bio-logicalfunctions
8–10
.Fewhavebeendemonstrated tohave phenotypicconsequences by loss-of-function experiments
6
. As a result, the func-tional role of lincRNA genes has been widely debated. Various pro-posals include that lincRNA genes act as enhancer regions, with theRNA transcript simply being an incidental by-product
8,9
, thatlincRNA transcripts act in
cis
to activate transcription
11
, and thatlincRNA transcripts can act in
trans
to repress transcription
12,13
.We therefore sought to undertake systematic loss-of-functionexperimentsonalllincRNAsknowntobeexpressedinmouseembry-onicstem(ES)cells
2,3
.EScellsarepluripotentcellsthatcanself-renein culture and can give rise to cells of any of the three primary germlayers including the germ line
14
. The signalling 
14
, transcriptional
15–17
and chromatin
15,18–21
regulatory networks controlling pluripotency have been well characterized, providing an ideal system to determinehow lincRNAs may integrate into these processes.Here we show that knockdown of the vast majority of ES-cell-expressed lincRNAs has a strong effect on gene expression patternsin ES cells, of comparable magnitude to that seen for the well-knownEScellregulatoryproteins.WeidentifydozensoflincRNAsthatuponloss-of-functioncauseanexitfromthepluripotentstateanddozensof additional lincRNAs that, although not essential for the maintenanceof pluripotency, act to repress lineage-specific gene expression pro-grams in ES cells. We integrate the lincRNAs into the molecularcircuitryofEScellsbydemonstrating that mostlincRNAs aredirectly regulatedbycriticalpluripotency-associatedtranscriptionfactorsand
,
30% of lincRNAs physically interact with specific chromatin regu-latory proteins to affect gene expression. Together, these resultsdemonstrate a regulatory network in ES cells whereby transcriptionfactors directly regulate the expression of lincRNA genes, many of which can physically interact with chromatin proteins, affect geneexpression programs and maintain the ES cell state.
lincRNAs affect global gene expression
To perform loss-of-function experiments, we generated five lentiviral-based short hairpin RNAs (shRNAs)
22
targeting each of the 226lincRNAs previously identified in ES cells
2,3
(see Methods and Sup-plementary Table 1). These shRNAs successfully targeted 147 lincRNAsand reduced their expression by an average of 
,
75% compared toendogenous levels in ES cells (see Methods, Fig. 1a, Supplementary Fig. 1 and Supplementary Table 2). As positive controls, we generatedshRNAs targeting 
,
50 genes encoding regulatory proteins, including bothtranscriptionandchromatinfactorsthathavebeenshowntoplacritical roles in ES cell regulation
17,20,23
; validated hairpins wereobtained against 40 of these genes (Supplementary Table 2). As nega-tive controls, we performed independent infections with lentivirusescontaining 27 different shRNAs with no known cellular target RNA.We infected each shRNA into ES cells, isolated RNA after 4days,and profiled their effects on global transcription by hybridization togenome-widemicroarrays(Fig.1a,seeMethods).Weusedastringentprocedure to control for nonspecific effects due to viral infection,generic RNA interference (RNAi) responses, or ‘off-target’ effects.Expression changes were deemed significant only if they exceededthe maximum levels observed in any of the negative controls, showedatwofoldchangeinexpressioncomparedtothenegativecontrols,andhadalowfalsediscoveryrate(FDR)assessedacrossallgenesbasedonpermutation tests (Fig. 1b, see Methods). This approach controls forthe overall rate of nonspecific effects by estimating the number and
*
These authors contributed equally to this work.
1
BroadInstituteofMITandHarvard,7CambridgeCenter,Cambridge,Massachusetts02142,USA.
2
DepartmentofBiology,MassachusettsInstituteofTechnology,Cambridge,Massachusetts02139,USA.
3
WhiteheadInstituteforBiomedicalResearch,9CambridgeCenter,Cambridge,Massachusetts02142,USA.
4
GenomicsResearchandDevelopment,AgilentTechnologies,SantaClara,California95051,USA.
5
Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
6
Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02114, USA.
0 0 M O N T H 2 0 1 1 | V O L 0 0 0 | N AT U R E | 1
Macmillan Publishers Limited. All rights reserved
 ©2011
 
magnitude of observed effects in the negative control hairpins, whereall effects are nonspecific.For 137 of the 147 lincRNAs (93%), knockdown caused a signifi-cant impact on gene expression (Supplementary Table 3), with anaverage of 175 protein-coding transcripts affected (range: 20–936)(Fig. 1c, Supplementary Fig. 2 and Supplementary Table 4). Theseresultsweresimilartothoseobtaineduponknockdownofthe40well-studied ES cell regulatory proteins: 38 (95%) showed significanteffects on gene expression, with an average of 207 genes affected(range: 28 (for Dnmt3l) to 1,187 (for Oct4)) (Fig. 1c, Supplemen-tary Fig. 2 and Supplementary Table 4). Although some individuallincRNAshavebeenfoundtoleadprimarilytogenerepression
12,13
,wefind that knockdown of the lincRNAs studied here largely led tocomparable numbers of activated and repressed genes (Supplemen-tary Fig. 2 and Supplementary Table 4). To assess off-target effectsfurther, we also profiled the effects of the second-best validatedshRNA targeting 10 randomly selected lincRNA genes. In all cases,secondshRNAsagainstthesametargetproducedsignificantlysimilarexpression changes (see Methods and Supplementary Table 5). Theseresults indicate that the vast majority of lincRNAs have functionalconsequences on overall gene expression of comparable magnitude(in terms of number of affected genes and impact on levels) to theknown transcriptional regulators in ES cells.
lincRNAs affect gene expression in
trans 
Following the observation that a few lincRNAs act in
cis
24,25
, somerecent papers have claimed that most lincRNAs act primarily in
cis
8,11,26
.Wefoundnoevidencetosupportthislatternotion:knockdownof only 2 lincRNAs showed effects on a neighbouring gene, only 13showed effects within a window of 10 genes on either side, and only 8showedeffectsongeneswithin300kb;theseproportionsarenogreaterthan observed for protein-coding genes (Supplementary Fig. 3 andSupplementary Table 6). In short, lincRNAs seem to affect expressionlargely in
trans
.Our results contrast with a recent study that concluded thatlincRNAs act in
cis
, based on the observation that knockdown of 7out of 12 lincRNAs affected expression of a gene within 300kb
11
. Theexplanationseemstobethatthethresholdintheprevious studyfailedto account for multiple hypothesis testing within the local region.Accounting for this, the effects on neighbouring genes are no greaterthan expected by chance and are consistent with our observationshere (see Methods).Although some lincRNAs can regulate gene expression in
cis
11,24,25
,determining the precise proportion of 
cis
regulators requires moredirectexperimentalapproaches.Wenotethatourresultsareconsistentwith observed correlations between lincRNAs and neighbouring genes
2,26
, which may represent shared upstream regulation
2,12
or localtranscriptional effects
10,27
. In addition, the lincRNAs studied hereshouldbedistinguishedfromtranscriptsthatareproducedatenhancersites
8,9
, the function of which has yet to be determined.
lincRNAs maintain the pluripotent state
We next sought to investigate whether lincRNAs have a role in regu-lating the ES cell state. Regulation of the ES cell state involves twocomponents: maintaining the pluripotency program and repressing differentiation programs
15
. To determine whether lincRNAs have aroleinthemaintenanceofthepluripotencyprogram,westudiedtheireffects on the expression of Nanog, a key transcription factor that isrequired to establish
28
and uniquely marks the pluripotent state
29,30
.We infected ES cells carrying a luciferase reporter gene expressedfrom the endogenous
Nanog 
promoter
31
with shRNAs targeting lincRNAs or protein-coding genes. We monitored loss of reporteractivity after 8days relative to 25 negative control hairpins acrossbiological replicates (see Methods). To ensure that the observedeffectswerenotsimplyduetoareductionincellviability,weexcludedshRNAs that caused a reduction in cell numbers (see Methods,Supplementary Fig. 4 and Supplementary Table 7). Altogether, weidentified 26 lincRNAs that had major effects on endogenousNanog levels with many at comparable levels to the knockdown of the known protein-coding regulators of pluripotency such as Oct4and Nanog (Fig. 2a and Supplementary Table 7). This establishes thatthese lincRNAs have a role in maintaining the pluripotent state.To validate further the role of these 26 lincRNAs in regulating thepluripotent state, we knocked down these lincRNAs in wild-type EScells and measured mRNA levels of pluripotency marker genes
Oct4
(also called
Pou5f1
),
Sox2
,
Nanog 
,
Klf4
and
Zfp42
after 8days. In allcasesweobservedasignificantreductionintheexpressionofmultiplepluripotency markers with
.
90% showing a significant decrease inboth
Oct4
and
Nanog 
levels(SupplementaryFig.5andSupplementary Tables8and9).Tocontrolforoff-targeteffects,westudiedadditionalhairpins targeting these lincRNAs. For 15 lincRNAs we had an effec-tive second hairpin. In all 15 cases, the second hairpin producedcomparable reductions in
Oct4
expression levels, showing that theobservations were not due to off-target effects (Fig. 2b and Sup-plementary Table 10). Notably,
.
90% of lincRNA knockdownsaffecting Nanog reporter levels led to loss of ES cell morphology (Fig. 2c and Supplementary Figs 6 and 7). Thus, inhibition of these26 lincRNAs lead to an increased exit from the pluripotent state.
lincRNAs repress lineage programs
To determine if lincRNAs act in repressing differentiation programswe compared the overall gene expression patterns resulting fromknockdown of the lincRNAs to published gene expression patternsresulting from induced differentiation of ES cells
32,33
and assessedsignificance using a permutation-derived FDR 
34
(see Methods).These states include differentiation into endoderm, ectoderm,neuroectoderm,mesodermandtrophectodermlineages.Asapositivecontrol for our analytical method, we confirmed the expected resultsthat the expression pattern caused by Oct4 knockdown was strongly associated with the trophoectoderm lineage
35
and the pattern causedby Nanog knockdown was strongly associated with endoderm differ-entiation
30
(Fig. 3a).
cb
Moxd1
linc1244
Ctgf 
95genes
   D   i   f   f  e  r  e  n   t   i  a   l   l  y  e  x  p  r  e  s  s  e   d  g  e  n  e  s
27 negative controlsKD
0 1    , 4   0   0   4   0   0   2   0   0   6   0   0   1    , 0   0   0   8   0   0   1    , 2   0   0   
Protein codinglincRNAOct4Stat3Klf4Sox20.0000.0040.0020.0010.003
   D  e  n  s   i   t  y
Differentially expressed genes
a
Select besthairpinIdentifyaffectedgenesKnock-downInfect withshRNAControlEmbryonicstem cellsControlhairpinslincRNAhairpinsNanog40%
Figure 1
|
Functional affects of lincRNAs.
, A schematic of lincRNAperturbationexperiments. ES cellsareinfected with shRNAs, knockdown levelis computed, the best hairpin is selected andprofiled on expression arrays, anddifferential gene expression is computed relative to negative control hairpins.
b
, Example of a lincRNA knockdown. Top: genomic locus containing thelincRNA. Bottom: heat-map of the 95 genes affected by knockdown of thelincRNA,expressionforcontrolhairpins(redline)andexpressionforlincRNAhairpins(blueline)areshown.
c
,Distributionofnumberofaffectedgenesuponknockdown of 147 lincRNAs (blue) and 40 well-known ES cell regulatory proteins (red). Points corresponding to five specific ES cell regulatory proteinsare marked.
RESEARCHARTICLE
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Macmillan Publishers Limited. All rights reserved
 ©2011
 
Using this approach, we identified 30 lincRNAs for which knock-downproducedexpressionpatternssimilartodifferentiationintospe-cific lineages (Supplementary Table 11). Among these lincRNAs, 13are associated with endoderm differentiation, 7 with ectoderm differ-entiation, 5 with neuroectoderm differentiation, 7 with mesodermdifferentiation and 2 with the trophectoderm lineage (Fig. 3a).Consistent with these functional assignments, we observed that most(
.
85%) of the 30 lincRNAs associated with specific differentiationlineages showed upregulation of the well-known marker genes forthe identified states
17,32
upon knockdown (such as
Sox17 
(endoderm),
Fgf5
(ectoderm),
Pax6 
(neuroectoderm), brachyury (mesoderm) and
Cdx2
(trophectoderm)) (Fig. 3b, Supplementary Figs 8 and 9 andSupplementary Tables 12 and 13).The fact that knockdown of these 30 lincRNAs induces geneexpression programs associated with specific early differentiationlineages indicates that these lincRNAs normally are a barrier to suchdifferentiation. Interestingly, most of the lincRNA knockdowns(
,
85%) that induce gene expression patterns associated with theselineages did not cause the cells to differentiate as determined by Nanog reporter levels (Supplementary Table 7) and Oct4 expression(Supplementary Fig. 10). This is consistent with observations forseveral critical ES cell chromatin regulators, such as the polycombcomplex; loss-of-function of these regulators similarly induceslineage-specific markers without causing differentiation
18,36,37
.Together, these data indicate that many lincRNAs have importantrolesinregulating the EScell state, including maintaining thepluripo-tent state and repressing specific differentiation lineages.
lincRNAs are targets of ES cell transcription factors
Having demonstrated a functional role for lincRNAs in ES cells, wesoughttointegratethelincRNAsintothemolecularcircuitrycontrol-ling the pluripotent state. First, we explored how lincRNA expressionis regulated in ES cells. Towards this end, we used published genome-wide maps of 9 pluripotency-associated transcription factors
16,38
and determined whether they bind to the promoters of lincRNAgenes. Of the 226 lincRNA promoters
,
75% are bound by at least 1of 9 pluripotency-associated transcription factors (including Oct4,Sox2,Nanog,c-Myc,n-Myc,Klf4,Zfx,SmadandTcf3)withamedianof 3 factors bound to each promoter (Fig. 4a, Supplementary Fig. 11andSupplementaryTable14),comparabletotheproportionreportedfor protein-coding genes
16
. Interestingly, the three core factors (Oct4,Sox2 and Nanog) bind to the promoters of 
,
12% of all ES celllincRNAs and
,
50% of lincRNAs involved in the regulation of thepluripotent state.TodetermineiflincRNAexpressionisfunctionallyregulatedbythepluripotency-associated transcription factors, we used shRNAs toknockdown the expression of 5 of the 9 pluripotency-associated tran-scription factor genes for which we could obtain validated hairpinsandprofiledtheresulting changesinlincRNAexpression after 4days.Upon knockdown of a transcription factor,
,
50% of lincRNA geneswhose promoters are bound by the transcription factor exhibitexpression changes (Fig. 4a); this proportion is comparable to that
ab
   O  c   t   4   N  a  n  o  g   l   i  n  c   1   2   5   3   l   i  n  c   1   3   3   1   l   i  n  c   1   5   5   7   l   i  n  c   1   5   6   2   l   i  n  c   1   6   0   2   l   i  n  c   1   2   3   0   l   i  n  c   1   3   3   5   l   i  n  c   1   4   6   3   l   i  n  c   1   5   8   2   l   i  n  c   1   6   1   2   l   i  n  c   1   3   0   4   l   i  n  c   1   3   5   6   l   i  n  c   1   4   0   0   l   i  n  c   1   4   5   6   l   i  n  c   1   4   9   0   l   i  n  c   1   5   1   7   l   i  n  c   1   5   8   8   l   i  n  c   1   6   1   7   l   i  n  c   1   6   2   3   l   i  n  c   1   6   2   7   l   i  n  c   1   6   3   3   l   i  n  c   1   3   8   8   l   i  n  c   1   3   9   0   l   i  n  c   1   5   2   6   l   i  n  c   1   5   5   2   l   i  n  c   1   4   3   4   l   i  n  c   1   3   0   7   l   i  n  c   1   6   0   4   l   i  n  c   1   4   7   0   l   i  n  c   1   2   4   2
EctodermNeuroectodermEndodermMesodermTrophectoderm012354
Sox17 
(endoderm)
Fgf5
(ectoderm)
Pax6
(neuroectoderm)
Brachyury 
(mesoderm)
Cdx2
(trophectoderm)1112 2.5
   L  o  g  -  e  x  p  r  e  s  s   i  o  n  c  o  m  p  a  r  e   d  w   i   t   h  c  o  n   t  r  o   l  s  s   h   O  c   t   4  s   h   N  a  n  o  g  s   h   l   i  n  c   1   2   3   0  s   h   l   i  n  c   1   2   5   3  s   h   l   i  n  c   1   5   6   2  s   h   l   i  n  c   1   6   0   2  s   h   O  c   t   4  s   h   N  a  n  o  g  s   h   l   i  n  c   1   2   3   0  s   h   l   i  n  c   1   3   3   5  s   h   l   i  n  c   1   4   6   3  s   h   l   i  n  c   1   5   5   2  s   h   O  c   t   4  s   h   N  a  n  o  g  s   h   l   i  n  c   1   3   8   8  s   h   l   i  n  c   1   3   9   0  s   h   l   i  n  c   1   4   5   6  s   h   l   i  n  c   1   5   8   8  s   h   O  c   t   4  s   h   N  a  n  o  g  s   h   l   i  n  c   1   3   8   8  s   h   l   i  n  c   1   3   9   0  s   h   l   i  n  c   1   4   3   4  s   h   l   i  n  c   1   5   2   6  s   h   O  c   t   4  s   h   N  a  n  o  g  s   h   l   i  n  c   1   4   7   0  s   h   l   i  n  c   1   6   0   4  s   h   l   i  n  c   1   6   2   3  s   h   l   i  n  c   1   6   2   7
Figure 3
|
lincRNAs repress specific differentiation lineages.
, Expressionchanges for each lincRNA compared to gene expression of five differentiationpatterns. Each box shows significant positive association (red, FDR 
,
0.01) forOct4 and Nanog (left) and for lincRNAs (right).
b
, Expression changes uponknockdownofOct4andNanog(blackbars)andrepresentativelincRNAs(grey bars) for five lineage marker genes. The expression changes (FDR 
,
0.05) aredisplayedonalogscaleasthe
-statisticcomparedtoapanelofnegativecontrolhairpins.
ac
   N  a  n  o  g  -   l  u  c   i   f  e  r  a  s  e   i  n   t  e  n  s   i   t  y
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0160,000120,000100,000140,00080,00060,00020,00040,000
NegativesProteinslincRNAs
shlinc1368shOct4shlinc1577shRFP NegativePositivelincRNAs
   B  r   i  g   h   t   O  c   t   4   D   A   P   I
b
   O  c   t   4
  r  e   l  a   t   i  v  e  e  x  p  r  e  s  s   i  o  n
00.20.40.60.811.2
      C     o     n      t     r     o      l     s      O     c      t      4      S     o    x      2      S      t     a      t      3      C      h      d      1      l      i     n     c      1      4      8      7      l      i     n     c      1      4      3      4      l      i     n     c      1      6      3      4      l      i     n     c      1      3      6      8      l      i     n     c      1      3      4      9      l      i     n     c      1      5      8      2      l      i     n     c      1      4      9      0      l      i     n     c      1      5      4      7      l      i     n     c      1      5      7      2      l      i     n     c      1      4      1      1      l      i     n     c      1      3      9      9      l      i     n     c      1      4      3      5      l      i     n     c      1      2      8      3      C     o     n      t     r     o      l     s      C      h      d      1      S     o    x      2      S      t     a      t      3      l      i     n     c      1      4      9      0      l      i     n     c      1      3      4      9      l      i     n     c      1      3      6      8      l      i     n     c      1      5      8      2      l      i     n     c      1      4      1      1      l      i     n     c      1      5      7      2      l      i     n     c      1      5      4      7      l      i     n     c      1      4      3      5      l      i     n     c      1      4      8      7      l      i     n     c      1      3      9      9      l      i     n     c      1      2      8      3      l      i     n     c      1      6      3      4      l      i     n     c      1      4      3      4
Hairpin 1Hairpin 2
Figure 2
|
lincRNAs are critical for the maintenance of pluripotency.
, Activity from a
Nanog 
promoterdriving luciferase, following treatment withcontrol hairpins (black) or hairpins targeting luciferase (green), selectedprotein-coding regulators (red), and lincRNAs (blue).
b
, Relative mRNAexpression levels of 
Oct4
afterknockdown of selectedprotein-coding(red)andlincRNA (blue) genes affecting Nanog-luciferase levels. The best hairpin(Hairpin1)andsecondbesthairpin(Hairpin2)areshown.Allknockdownsaresignificant with a
-value
,
0.01. Error bars represent standard error (
n
5
4).
c
, Morphology of ES cells and immunofluorescence staining of Oct4 for anegativecontrolhairpin(blackline)andhairpinstargetingOct4(redline),andtwo lincRNAs (blue line). The first row shows bright-field images, the secondrowshowsimmunofluorescencestainingoftheOct4protein,andthethirdrow shows DAPI staining of the nuclei.
ARTICLERESEARCH
0 0 M O N T H 2 0 1 1 | V O L 0 0 0 | N AT U R E | 3
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